The mechanism of thioredoxin reductase from human placenta is similar to the mechanisms of lipoamide dehydrogenase and glutathione reductase and is distinct from the mechanism of thioredoxin reductase from Escherichia coli

Abstract

Thioredoxin reductase, lipoamide dehydrogenase, and glutathione reductase are members of the pyridine nucleotide-disulfide oxidoreductase family of dimeric flavoenzymes. The mechanisms and structures of lipoamide dehydrogenase and glutathione reductase are alike irrespective of the source (subunit M(r) approximately 55,000). Although the mechanism and structure of thioredoxin reductase from Escherichia coli are distinct (M(r) approximately 35,000), this enzyme must be placed in the same family because there are significant amino acid sequence similarities with the other two enzymes, the presence of a redox-active disulfide, and the substrate specificities. Thioredoxin reductase from higher eukaryotes on the other hand has a M(r) of approximately 55,000 [Luthman, M. & Holmgren, A. (1982) Biochemistry 21, 6628-6633; Gasdaska, P. Y., Gasdaska, J. R., Cochran, S. & Powis, G. (1995) FEBS Lett 373, 5-9; Gladyshev, V. N., Jeang, K. T. & Stadtman, T.C. (1996) Proc. Natl. Acad. Sci. USA 93, 6146-6151]. Thus, the evolution of this family is highly unusual. The mechanism of thioredoxin reductase from higher eukaryotes is not known. As reported here, thioredoxin reductase from human placenta reacts with only a single molecule of NADPH, which leads to a stable intermediate similar to that observed in titrations of lipoamide dehydrogenase or glutathione reductase. Titration of thioredoxin reductase from human placenta with dithionite takes place in two spectral phases: formation of a thiolate-flavin charge transfer complex followed by reduction of the flavin, just as with lipoamide dehydrogenase or glutathione reductase. The first phase requires more than one equivalent of dithionite. This suggests that the penultimate selenocysteine [Tamura, T. & Stadtman, T.C. (1996) Proc. Natl. Acad. Sci. USA 93, 1006-1011] is in redox communication with the active site disulfide/dithiol. Nitrosoureas of the carmustine type inhibit only the NADPH reduced form of human thioredoxin reductase. These compounds are widely used as cytostatic agents, so this enzyme should be studied as a target in cancer chemotherapy. In conclusion, three lines of evidence indicate that the mechanism of human thioredoxin reductase is like the mechanisms of lipoamide dehydrogenase and glutathione reductase and differs fundamentally from the mechanism of E. coli thioredoxin reductase.

Figure 1

Phylogenetic tree. The distances between branch points are not intended to reflect time quantitatively. A gene sequence from the worm (Caenorhabditis elegans) is homologous with the sequence of hTrxR (1).

Figure 2

Titration of oxidized hTrxR with sodium dithionite. (A) Representative spectra showing the emergence of the EH₂ species during the first phase of titration. Spectra are in order from solid line to dotted: 18 μM of oxidized enzyme before addition of dithionite, then 0.9, 1.5, 2.1, and 2.7 eq. (B) Representative spectra showing the emergence of the EH₄ species during the second phase of titration. Spectra start with the solid line; equivalents of dithionite added are: 2.8, 3.2, 3.5, 4.0, and 4.4. Note the 0.1 equivalent of methyl viologen radical in the last spectrum with the two major absorbance bands, 394 and 600 nm. (Inset) Observed absorbance changes upon titration of hTrxR with sodium dithionite. Conditions are as in Materials and Methods. Note the equivalent points of 0.9, 1.9, and 2.8 where several data points are shown representing longer equilibration times of 50, 50, and 40 minutes, respectively. For all other data points, spectra were recorded after 3–7 min of equilibration time. A_{540 nm} (closed circles) = 0.02 and A_{463 nm} (open squares) = 0.05 for each marking on the left and right y axes, respectively.

Figure 3

Rapid reaction of hTrxR with NADPH. Anaerobic reduction of 8 μM of enzyme with 5 eq of NADPH (after mixing). Times in milliseconds for each spectrum are as follows: 1, oxidized; 2, 9; 3, 31; 4, 63; and 5, 415. (Inset) Rapid reaction kinetic traces observed at 463 and 540 nm for the reduction of hTrxR by 5 eq of NADPH. Observed traces with fits to a sum of three exponentials. A_{540 nm} = 0.06–0.10 (0.01 for each marking) on the left axis and A_{463 nm} = 0–0.04 (0.01 for each marking) on the right y axis.

Figure 4

Inactivation of two electron-reduced hTrxR by BCNU. HTrxR (1.6 units/ml; corresponding to 1 nmol subunit/ml) was incubated at 37°C in hTrxR assay buffer (100 mM potassium phosphate/2 mM EDTA, pH 7.4) with various concentrations of BCNU in the presence of 100 μM of NADPH (control without NADPH, closed circles). Aliquots were diluted 100-fold and assayed for residual activity. The apparent half-times of inactivation and BCNU concentrations are: open circles, 1.7 min, 1 mM; closed triangles, 5 min, 500 μM; and closed squares, 17 min, 100 μM. Because the decomposition of BCNU to chloroethyl isocyanate and other reactive species (t_1/2 = 58 min under these conditions) is often rate-limiting (32), the kinetics of EH₂ inactivation can be more complex than suggested by this figure.